Dietary supplements are used by consumers to prevent and to treat prostate cancer. Recently, an animal prostate cancer model has allowed the safety and efficacy of these supplements to be tested. We have established colonies of TRansgenic Adenocarcinoma of the Mouse Prostate (TRAMP) mice, estrogen receptor-deficient (ERalphaKO and ERbetaKO) mice, and ER-deficient mice expressing the TRAMP transgene. Thus, a systematic approach is now possible for the identification of molecular mechanisms through which botanicals affect tumor incidence and progression. Exciting data from our model clearly show that ERalphaKO mice are highly protected against PDC (poorly differentiated carcinoma) of the prostate and ERbetaKO mice are highly susceptible to PDC. In addition to estrogen-signaling, two more signaling pathways are also believed important in mediating the initiation and progression of prostate cancer: DNA methylation and antioxidant- signaling. Our overall hypothesis is that differential regulation of ERalpha and ERP by plant phytoestrogens, acting essentially as natural SERMs, will be effective in human prostate cancer prevention and treatment. Our goals are to characterize responses of key prostate tumor biomarkers to botanicals and to provide novel molecular mechanisms for these responses. Aim la: We will use the TRAMP mouse model to perform in vivo cancer prevention trials with 2 high priority botanicals (spinach and green tea;and their bioactive markers/components) using two doses in ER WT or ER-minus / TRAMP mice to investigate the in vivo roles of ERalpha and ERbeta proteins in prostate tumorigenesis and their impact on the development of PDC. Aim Ib: We will confirm our previous findings that in TRAMP mice the development of PDC of the prostate is promoted by ERalpha and prevented by ERbeta using ERalpha and ERbeta specific ligands. Aim 2: We will profile the molecular mechanisms by which the same 2 priority botanicals and their bioactive markers plus genistein induce the effect on selected intracellular DNA methylation, estrogenic and antioxidant pathways in both human and mice cultured prostate tumor cells. Finally, in Aim 3: We will confirm in vivo in mouse tissues from Aim #1, the role of selected botanicals and their bioactive markers in regulation of cellular pathways of importance to carcinogenesis, including selected intracellular DNA methylation, estrogenic and antioxidant pathways. We will also use already collected genistein-treated tissue samples because of genistein's reported effect on PDC and our finding of its effect on WDC (well differentiated carcinoma). These studies will provide fundamental insights into prostate tumor biology, and assess the role of these botanicals in modifying the incidence and progression of prostate tumors in humans.